The present invention relates to methods of generating and isolating proliferative, non terminally differentiated connective tissue progenitor cells from embryonic stem cells and embryoid bodies and, more particularly, to methods of using such cells for cell based therapy and tissue engineering applications.
Cell-based tissue engineering is an evolving interdisciplinary area that offers new opportunities for clinical applications, creating a tool for repairing and replacing damaged or lost tissues with biological substitutes. The shortage of organ transplants and the exceeding number of patients on waiting lists greatly encourage the development of this field. The fundamentals of tissue engineering combine cells, bioactive matrices and chemically and biophysically defined in-vitro culture conditions. For tissue engineering, cells must be easily isolated, sufficient in numbers, with a great proliferation capacity and a well-defined differentiation potential. A number of cell sources have been suggested including primary cells and stem cells which are either host- or donor-derived. A wide array of matrices, either biologically or synthetically designed, are to provide the mechanical cues and three-dimensional environment, supporting cell attachment, migration, proliferation, differentiation and organization into complex tissues. Controlling stem cell proliferation and differentiation into any desired cell type requires the identification of chemicals (e.g., hormones and growth factors) and/or growth conditions (e.g., static or dynamic culturing conditions), which regulate the differentiation into the desired cell or tissue.
Connective tissue repair and regeneration are subjected to intensive research within clinical medicine. Damaged or disordered connective tissues, such as bone, cartilage and tendons need to be reconstructed or replaced due to traumatic injuries, degenerative diseases, tumor resections and congenital malformations. Current strategies in reconstructive orthopedic surgery include the use of autografts, allografts and artificial substitutes, all subjected to various limitations. While the use of cell grafts is limited by availability and morbidity, synthetic grafts are osteoconductively inferior to their biological counterparts, and could fail.
Mesenchymal stem cells (MSCs) have previously been derived from bone (Sottile, V et al 2002), bone marrow (Pittenger, M. F et al, 1999), muscle (Mastrogiacomo, M et al 2005), and fat (Zuk, P. A et al, 2001), and were capable of differentiating into adipocytic, chondrocytic, osteocytic or myogenic lineages.
Human embryonic stem cells (hESCs) hold great promise as a source of cells for tissue engineering. Their ability for practically unlimited self-renewal can potentially provide the required amount of cells needed for ex vivo tissue construction. In addition, they are characterized by a developmental potential to differentiate into any cell type of the mammalian embryo, and recently have been efficiently derived by means of somatic cell nuclear transfer, creating patient-specific immune-matched cell lines. hESCs have been shown to be able to form vascularized tissue-like structures when grown on either PLGA/PLLA or alginate porous scaffolds.
Several approaches have been recently described for isolating MSC-like cells from hESCs.
For example, Olivier E N., et al., 2006 [Olivier, E. N., et al., 2006, Stem Cells 24, 1914-1922] cultured spontaneously differentiating cells of hESCs colonies which were scraped from the edges of the colonies (“raclures”) until a thick multi-layer epithelium was formed (at least 4 weeks). The cells of the thick epithelium were further dissociated and routinely passaged. The resulting cells exhibited surface phenotype of MSCs such as CD105+/CD166+/HLA-ABC+/CD73+/CD45−/HLA-DR− and were capable of in-vitro differentiation into osteoblasts and adipocytes. However, the use of such a method (the “raclure method”) is limited because specific ESCs are mechanically scraped from ESC colonies cultured on mouse feeder cells, which may result in a crude, non-defined, population of cells.
In another study Barberi, T., et al. (2005) co-cultured hESCs on mouse OP-9 stromal feeder layers and following 40 days of co-culture isolated CD73-positive cells (MSC-like cells) and replated them in the absence of the stromal cells. However, this method is limited by the extremely low yield of the MSC-like cells (only 5% of the cells were CD73-positive cells) and by the co-culturing of the hESCs on mouse feeder-layers, which complicates culturing procedures and limits the use for cell-based therapy.
Other approaches utilized ESCs which have undergone spontaneous differentiation to embryoid bodies (EBs) in order to generate in-vitro committed cells of the osteogenic lineage.
For example, EBs were dissociated into single cells and were further induced to terminally differentiate into the osteogenic lineage by culturing them in an osteogenic medium without passaging for 21 (Sottile V, et al., 2003) or 28 (Bielby et al., 2004) days. The resulting cells expressed osteogenic markers and formed mineralized nodules.
Other studies obtained committed cells of the osteogenic lineage by plating intact EBs on adherent culture plates and culturing the EBs for at least 22 days without passaging (Cao T., et al. 2005). Thus, Cao et al. (2005), Bielby et al. (2004) and Sottile et al. (2003) concluded that culturing cells of EBs in an osteogenic medium results in terminally differentiated cells of the osteoblast cell lineage.
There is thus a widely recognized need for, and it would be highly advantageous to have, hESC-derived multipotent cells for tissue engineering devoid of the above limitations.